Insulin is the major anabolic hormone promoting uptake and storage of molecules, including glucose, amino acids and fatty acids (1). Insulin acts through a cell surface tyrosine kinase receptor that phosphorylates downstream substrates. Tyrosine phosphorylation of substrates, such as the Insulin Receptor Substrate (IRS) family of scaffold proteins, serves to recruit SH2 domain containing proteins, including PI 3-kinase, leading to propagation of insulin signalling cascades (1). Whilst many of the molecules that act downstream of PI 3-kinase are known, such as the Ser/Thr kinases Akt and atypical PKCs, knowledge of the signalling pathways is incomplete (1). In an effort to address this, a proteomic screen was performed to identify novel insulin-regulated phosphoproteins that may act downstream of PI 3-kinase in 3T3-L1 adipocytes (2).
Inosine-5′ monophosphate dehydrogenase (IMPDH) is a highly conserved, ubiquitously expressed enzyme, which catalyses a key step in the de novo biosynthesis of guanine nucleotide, the NAD-dependent conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) (3). In mammals there are two IMPDH isoforms, termed type I and type II, that show 84% amino acid identity and exist as homotetramers (4, 5). Increased IMPDH activity is associated with proliferation and malignancy, an observation that was recognised almost thirty years ago (6). As such, IMPDH represents a principal target for therapeutic intervention in anti-neoplastic and immunosuppressive treatments and has been the subject of intensive biochemical research (7). Recently mutations in IMPDH type I have been shown to cause the RP10 form of the degenerative retinal disorder autosomal dominant retinitis pigmentosa (adRP) in humans (8, 9).
Lipid bodies facilitate storage of intracellular lipid in mammalian cells (10). They are ubiquitous, ranging in size from 50 nm to 100 μm, and are made up of a neutral lipid core of diacylglycerol, triacylglycerol (TAG) and sterol ester surrounded by a phospholipid monolayer. Several proteins have been shown to associate with lipid bodies, the best characterised being adipocyte differentiation-related protein (ADRP) or adipophilin (the latter will be referred to as adipophilin to prevent confusion with adRP) (11, 12) and perilipin (13, 14) which form a proteinaceous coat around the lipid bodies (15). Treatment of cells with oleic acid, which is used as a substrate for TAG synthesis, promotes accumulation of lipid bodies and has recently been shown to be sufficient to promote translocation of several proteins to these structures (16-19). Whilst current understanding of the biogenesis of lipid bodies is incomplete they are believed to form between the two leaflets of the Endoplasmic Reticulum (ER), prior to budding off into the cytoplasm (20).
In work leading up to the present invention, it was discovered that IMPDH is a target of the insulin signal transduction pathway and a novel lipid body associated protein. The present inventor also found that insulin stimulates phosphorylation and translocation of IMPDH to lipid bodies, the sites of intracellular lipid storage, and both events are blocked by inhibition of PI 3-kinase. By contrast, oleate was shown to stimulate robust translocation of IMPDH to lipid bodies in the absence of detectable phosphorylation although this translocation was found to be sensitive to PI 3-kinase inhibition.
In addition, it was shown that inhibition of oleate induced IMPDH translocation correlates with decreased lipid accumulation. It was also shown that inhibition of IMPDH translocation correlates with a reduction in lipid accumulation (by ≧60%), that IMPDH protein expression is markedly (≧10 fold) and transiently increased during the differentiation of preadipocytes (both 3T3-L1 and primary human preadipocytes), but not other cell types such as myoblasts, and that the period of maximal IMPDH expression correlates with the period of lipid body accumulation.
Further, it was established that platelet derived growth factor (PDGF) stimulates IMPDH phosphorylation in 3T3-L1 fibroblasts, and this was shown to be dependent upon PI 3-kinase activation. This indicated, therefore, that the PI 3-kinase dependent phosphorylation of IMPDH is not specific to insulin, but may represent a common effect of agonists that are able to promote activation of PI 3-kinase. PDGF has recently been shown to promote increased synthesis of membrane lipids, and this also occurs through a PI 3-kinase dependent pathway (51).
From the foregoing, the present inventor proposed that adipogenesis or lipid accumulation can be modulated in vivo by modulating the level or functional activity (e.g., enzymic activity or level of phosphorylation) of IMPDH. This proposal has been reduced to practice in molecules and methods for modulating the level or functional activity of IMPDH as means of inhibiting or promoting adipogenesis in adiposity-related conditions, or for diagnosing predisposition to obesity, as described hereinafter.